Regulatory mechanisms responsible for limiting thrombus development are critical for maintaining normal hemostasis. New findings showing that TFPI? binds coagulation factor V(a) and inhibits prothrombinase assembly highlights that out understanding of the initiation of coagulation is evolving. Work from my laboratory has laid the groundwork for deciphering the mechanistic bases that may underpin how TFPI? mediates these anticoagulant effects. We hypothesize that through molecular mimicry, TFPI? uses key structural regions of the FV B-domain that remain following partial cleavage of the procofactor during the early phases of thrombus development. These include basic (BR) and acidic (AR) regions that are key auto-inhibitory elements responsible for keeping FV in an inactive procofactor state. The objectives of this proposal are to decipher these molecular processes, examine how they influence FV(a) activation and function, provide evidence its biologically relevant and identify which pool of FV (plasma or platelet-derived) contributes to these effects. A further goal is to better understand the molecular bases by which TFPI? modulate naturally occurring forms of FV including FV-East Texas and FV-Amsterdam, recently identified spliced forms of FV found in plasma that are missing a large portion of the B-domain. These new angles of thinking about FV activation, its biology, and regulation opens up several unexplored lines of experimentation. In the first aim, we will characterize the structural/functional determinants that maintain the FV procofactor state and determine whether TFPI? employs these molecular surfaces to dampen cofactor function. New biochemical and structural approaches (via collaborations) will be pursued to provide detailed insight into these molecular interactions. In the second aim, we will investigate the biochemical properties of naturally occurring forms of FV that lack the BR and characterize their interaction with TFPI?. We hypothesize that TFPI? utilizes a different set of structural determinants to bind different forms of FV(a) and the comple mediates its anticoagulant effects through multiple mechanisms. In the last aim, we will address the physiologic relevance of the FVAR-TFPI? interaction, determine how it may regulate thrombus development in vivo, and investigate whether this is mediated by plasma and/or platelet forms of FV. We hypothesize partially cleaved forms of FV are abundant at the site of the developing thrombus and that they are targets for TFPI?. We speculate that disruption of this regulatory mechanism results in altered kinetics of thrombus growth. We will test these innovative ideas using biochemical, kinetic, biophysical, structural and in vivo approaches employing a host of custom FV reagents. Knowledge gained from this proposal will move the field forward by providing an exceptional level of molecular detail on the structural and functiona regulation of FV released at the site of vascular injury. Because FV/Va has such a profound impact on thrombin generation, studies in this application will have important implications in developing new ways to modulate hemostasis to control bleeding or limit thrombosis.